T-lymphotropic retrovirus monoclonal antibodies

ABSTRACT

The present invention relates to monoclonal antibodies, cell lines producing the monoclonal antibodies, assays using the antibodies for the detection of HIV-1 and HIV-2 gene products, and diagnostic kits. More particularly, the monoclonal antibodies react with the p24/p26 capsid protein. Assays using monoclonal antibodies that can simultaneously detect HIV-1 and HIV-2 are disclosed.

This is a continuation of U.S. Ser. No. 08/044,340 filed Apr. 7, 1993,now abandoned, which is a divisional of U.S. Ser. No. 07/825,447, filedJan. 23, 1992, now U.S. Pat. No. 5,210,181, which is a file wrappercontinuation of U.S. Ser. No. 07/351,882 filed May 15, 1989, nowabandoned.

FIELD OF THE INVENTION

The invention relates to monoclonal antibodies, peptides that comprisethe epitopes of said monoclonal antibodies and assays utilizing saidmonoclonal antibodies and said peptides for the detection ofT-lymphotropic retroviruses, particularly HIV-1, HIV-2 and SIV.

BACKGROUND OF THE INVENTION

The T-lymphotropic retrovirus family includes among other lentivirusesthe simian retrovirus SIV and the human retroviruses HIV-1 (the likelyetiologic agent of AIDS) and HIV-2. Although HIV-1 and HIV-2 are relatedevolutionally, nucleic acid sequence analysis reveals that HIV-2 is moreclosely related to SIV than it is to HIV-1. Guyader et al. (1987) notedonly 42% overall genomic sequence identity between the HIV-1 and HIV-2isolates they compared. Patients infected with HIV-2 can manifestdisorders that typify AIDS, purely neurologic disease or asymptomaticinfections (Kuhnel et al., 1988) despite HIV-1-related ultrastructuraland biological properties such as in vitro cytopathogenicity and CD4tropism (Clavel et al., 1986).

The HIV-1 and HIV-2 genomes have a typical retroviral configurationcomprising LTR's, gag and env regions that encode viral structuralproteins, sequences encoding one or more enzyme, including a reversetranscriptase and other ORF's and regulatory elements. The gag region ofHIV-1 encodes a precursor peptide known as p55. p55 is processed toproduce among other proteins the major core or capsid protein known asp24. In HIV-2, the analogous E precursor is larger, known as p57, andthe major core protein is known as p26. Although a high degree ofconservation of the gag proteins of HIV-1 and HIV-2 was expected,Guyader et al. (1987) found only 58% identity of amino acids betweenHIV-1 and HIV-2 gag proteins. Even among distant isolates of HIV-1 thereis a greater than 90% identity of gag proteins. That and other datasupport the hypothesis that although HIV-1 and HIV-2 are somewhatrelated, they are nevertheless distinct retroviral species.

Because HIV-1 and possibly HIV-2 have such an impact on the human immunesystem, it is desirable, in fact imperative that sensitive, rapiddiagnostic assays for detecting presence of HIV be available forpopulation screening, quality control in blood banks, diagnosis,furtherance of our understanding of those viruses to assure the goal ofobtaining a vaccine and cure, and the like. Because of ease andconvenience, it is preferable that the assays be immunology-based, suchas ELISA's, and for reproducibility, specificity and consistency thatthe reagents be monoclonal antibodies and defined antigenic peptides.Because p24 antigenemia has been shown to be an early sign of HIVinfection (Kessler et al., 1987; Wall et al., 1987) and the observationthat clinical progression of AIDS sequelae is associated with reductionin anti-p24 while patients with AIDS can die with high levels ofanti-env titers (Coates et al., 1987), it would be advantageous for theassay to be directed to detecting gag products such as p24/p26.

Weiss et al. (1988) identified human serum samples that containedantibodies specific to HIV-2 gp130 in radioimmunoprecipitation assaysand in ELISA's. Those antibodies showed low level HIV-1 crossreactivityin a VSV pseudotype neutralization assay and in a neutralization ofC8166 syncytia formation assay.

Minassian et al. described a monoclonal antibody identified as R1C7 thatwas raised against HIV-2. R1C7, an anti-capsid antibody (p26), reactednot only with the three HIV-2 isolates tested, but with the five HIV-1isolates and seven SIV isolates that were tested. In immunoblots, R1C7bound to 55KD and 26KD HIV-2 proteins, to 24KD and 55KD HIV-1 proteinsand to a 28KD SIV protein.

Niedrig et al. developed a panel of 29 monoclonal antibodies to HIV-1.One antibody was directed to p17 and its precursor p32 whereas theremainder reacted with p24 and some of those also reacted with p55. Thep17 antibody was found to be HIV-1 specific. Of the 28 anti-p24antibodies, 20 reacted in immunoblots with the corresponding capsidprotein (p26) of HIV-2 and five of those also recognized thecorresponding SIV protein, p28. Niedrig et al. make no mention ofantibody titer, the efficacy of the antibodies in a antigen captureassay or which of the antibodies bind to p26, p55 or both. Furthermore,several of antibodies reacted with a 22KD protein of unknown function inHIV-2 preparations.

Many diagnostic kits and assays have been developed for the detection ofHIV-1 in samples of sera, blood, blood products or other body tissues.The assays use a variety of techniques such as Western blot,enzyme-linked immunosorbent assay (ELISA) or indirect immunofluorescentassay and employ either antibodies to whole virus or purified viralantigens, see for example, Gallo et al., U.S. Pat. No. 4,520,113;Sarngadharan, et al., (1984); and Robert-Guroff et al. (1982).

SUMMARY OF THE INVENTION

The instant invention relates to monoclonal antibodies, the cell linesproducing those antibodies, the peptides that comprise the epitopes ofthose antibodies and assays using those antibodies and peptides for thedetection of HIV-1 and HIV-2 gene products as well as SIV gene products.In particular, the antibodies react with the p24/p26 capsid protein. Thenonapeptide that comprises an HIV-1 /HIV-2 conserved epitope isdisclosed and a capture ELISA using a combination of three monoclonalantibodies that can detect simultaneously HIV-1 and HIV-2 is disclosed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Graph depicting reactivity of culture supernatants in captureELISA. A detailed legend appears in Table 1.

FIGS. 2a and 2b. Photographs of immunoblot nitrocellulose stripsdetermining the specificity of anti-HIV antibodies.

FIG. 3. Protein A-purified antibodies were used as probe to separatedHIV-2 proteins in immunoblots. Lanes 1 and 2 are positive controls andLane 3 is a negative control.

FIG. 4. Diagram of some of the recombinant p24 peptides used to mapepitopes.

FIG. 5. Diagram of four regions of p24 to which various monoclonalantibodies bind.

FIGS. 6a and 6b Photographs of Westerns reacting various monoclonalswith blotted gag and gag fragments. Lane 1 in each photo contains wholevirus lysate. Lane 5 in each photo is a negative control p24⁻ plasmidand Lane 6 in each photo is another negative control containingnon-HIV-infected MOLT lysate.

FIG. 7. Graph representing results of ELISA's using sequentialoverlapping nonapeptides as antigen to determine epitope of 7-D4.

FIG. 8. Diagram depicting epitope mapping using sequential overlappingnonapeptides as antigen in ELISA.

FIG. 9. Composition of the regions that comprise the 7-D4 epitope.

FIG. 10. Graph of sensitivity of a capture ELISA using two anti-p24antibodies, 6-C10 and 5-B4, on the solid phase and HIV-1 infected MOLT 3lysate as the antigen. An HRP conjugated human anti-HIV was thereporter.

FIG. 11. Graph of sensitivity of a capture ELISA using 6-C10 and 5-B4with and without 7-D4 on the solid phase to detect p26 of HIV-2.

FIG. 12. Dose response curve for HIV-1 and HIV-2 in a capture ELISAusing 6-C10, 5-B4 and 7-D4.

FIG. 13. Comparison of HIV-1 dose response curves between the threeantibody capture ELISA and a reverse transcriptase assay.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention relates to monoclonal antibodies and theirproduction, immunoassays and oligopeptides. The methods that were usedare known in the art and are discussed only briefly throughout thespecification. Suitable methods to practice the invention may be foundin Meth Enzymology 121, (1986) and other available reference materials.

Preparation of Monoclonal Antibodies

Monoclonal antibodies were produced according to established procedures(Kohler & Milstein, 1975). Briefly, female BALB/c mice were immunizedintraperitoneally repeatedly with lysates of HIV-1 infected MOLT 3 cellsemulsified in complete Freund's adjuvant (50%). Sensitized spleen cellswere fused with P3X63-Ag8.653 myeloma cells using PEG 1500.Heterokaryons were selected in HAT medium, cloned and screened forreactivity to HIV antigens in a capture ELISA. The IgG fraction ofpolyclonal human anti-HIV was coated onto wells of microtiter dishes.HIV-1 (produced in MOLT 3 cells) and culture supernatants were addedsimultaneously to the wells. Bound murine antibodies were detected withan enzyme-labelled anti-mouse Ig antibody. Data representative of thescreening is depicted in FIG. 1. Designation of the sample numbers isset forth in Table 1.

                  TABLE 1    ______________________________________    ELISA screening of Fusion F86    Sample No.   Designation    ______________________________________    1            5-B4    2            5-D9    3            5-E2    4            5-F12    5            6-B9    6            4-E6    7            6-C10    8            6-E11    9            6-F6    10           10-B2    11           10-C12    12           10-D1    13           2-C8    14           7-D4    15           7-E1    16           7-E10    17           7-F3    18           8-E7    19           9-B7    20           9-D5    21           F86 Bleedout*    22           NMS**    23           Negative Control    ______________________________________     *Serum obtained at sacrifice     **(Normal Mouse Serum

The hybridoma producing the monoclonal antibody designated 7D4 wasdeposited at the American Type Culture Collection, Rockville, Md, USA,under the terms of the Budapest Treaty, and was given an identificationnumber of HB 11254.

Western Blots

Candidate anti-HIV clones were tested further in Western blots (Towbinet al., 1979). Lysates of HIV-infected MOLT 3 cells were separatedthrough a 12% acrylamide gel under denaturing conditions. The proteinswere transferred to nitrocellulose and individual strips were blockedand reacted with the culture supernatants. Bound antibody was detectedusing an enzyme-labelled goat anti-mouse Ig antibody. Antibodiesreacting specifically with p24 were selected (FIG. 2). Designation ofthe strips is set forth in Table 2.

                  TABLE 2    ______________________________________    Western Blot Analysis of Anti-p24 mAbs    Strip #     Designation    ______________________________________    1           Positive Control    2           5-B4    3           5-D9    4           5-E2    5           5-F12    6           Positive Control    7           6-B9    8           6-C9    9           6-C10    10          6-E11    11          Positive Control    12          6-F6    13          10-B2    14          10-C12    15          Positive Control    16          10-D1    17          10-H1    18          7-D4    19          Positive Control    20          7-E1    21          7-E10    22          7-F3    23          8-E7    24          Positive Control    25          9-B7    26          9-D5    ______________________________________

The anti-p24 antibodies were then tested for cross-reactivity to p26 ofHIV-2 in immunoblots. HIV-2 lysates were separated, blotted and reactedwith the anti-p24 antibodies. Two antibodies, 7-D4 and 5-D9 reactedstrongly with p26 (FIG. 3). Designation of the strips is set forth inTable 3.

                  TABLE 3    ______________________________________    Cross-Reactivity of Anti-p24 mAbs    with p26 of HIV-2    Strip       mAb    ______________________________________    1           Hu-anti-HIV-1 IgG    2           OSS 39-B-3    3           MOPC 21 (IgG1)    4           F86/          5-B4    5                         5-D9    6                         6-C10    7                         7-E10    8                         5-E2    9                         9-B7    10                        7-F3    11                        6-F6    12                        9-D5    13                        7-D4    14                        6-E11    15                        7-E1    ______________________________________

In a related experiment, 7-D4 recognized a protein of approximately27,000 molecular weight in lysates of SIV_(MAC).

Epitope Mapping

The amino acids that comprise the p24 epitope of 7-D4 were mapped in thefollowing manner. The gag region and portions of gag were subcloned inan expression vector. Briefly, viral DNA of a λ_(HAT) bacteriophage(cDNA library HIV-1_(RF) , clone HAT 3 (Starcich et al., (1986)) wasdigested with EcoRI and by ligation into the pBR322-derived plasmidpMLB1113 to produce a plasmid identified as clone 29 which contained theEcoRI/SstI gag/pol ORF. Clone 29 was digested with SstI to removeextraneous vector sequences and religated to produce plasmid gag/pol1.2. This latter plasmid was sonicated, blunt-ended and ligated withEcoRI linkers. The mixture was then digested with EcoRI, ligated intoλORF8 (Meissner et al. 1987) and packaged. A λORF8 expression librarywas generated in E. coli and screened with a human anti-HIV polyclonalantibody and a mouse anti-p24 (HIV-1) monoclonal antibody. The positiveswere selected, expanded and the expressed peptides were characterized byWestern blotting, immunoassay and nucleotide sequencing. The recombinantp24 peptides gag 8, gag 126, gag 107 and gag 141 were expressed in E.coli. Separately, clone 29 was used as a template and oligonucleotidescorresponding to the 5' and 3' ends of the published sequence were usedin a polymerase chain reaction to generate a complete sequence of thegag protein p24. The 5' end contained an EcoRI site and the 3' endcontained a BamHI site. The reaction product was digested with EcoRI andBamHI and then ligated into pMLB1113. A recombinant p24 protein, gag24.5, was expressed in E. coli. The characterization of the recombinantp24 peptides is presented in FIG. 4.

The various recombinant p24 peptides were used as antigen in ELISA's andin Western blots to determine whether or not a given monoclonal antibodybound a given peptide. The reactivity pattern of any one monoclonalantibody with the panel of p24 peptides allowed a localization of therecognized epitope to one of four regions as shown in Table 4 and FIGS.5 and 6.

                  TABLE 4    ______________________________________    Immunochemical Analysis of Anti-24 mAbs    Using Recombinant Peptides             gag    gag      gag  gag    gag  mAb    mAb      24.5   8        126  107    141  group    ______________________________________    5-B4     +      -        +    -      -    B    5-D9     +      +        +    -      -    C    5-E2     +      +        -    -      -    D    5-F12    +      -        +    -      -    B    6-C10    +      +        +    -      -    C    6-E11    +      -        -    -      -    A    6-F6     +      -        -    -      -    A    7-D4     +      -        +    -      -    B    7-E1     +      -        +    -      -    B    7-E10    +      +        -    -      -    D    7-F3     +      -        +    -      -    B    8-E7     +      -        -    -      -    A    9-B7     +      +        -    -      -    D    9-D5     +      -        -    -      -    A    10-B2    +      -        +    -      -    B    10-C12   +      -        -    -      -    A    ______________________________________

Because 7-D4 bound only to gag 24.5 and gag 126, it was possible todeduce that the 7-D4 epitope mapped to region B delimited by amino acidresidues 142-209.

To further localize the epitope of 7-D4, synthetic sequentialoverlapping nonapeptides were made for the B region of p24. Eachnonapeptide served as the solid phase antigen in a series of ELISA's todetermine maximal binding affinity of the monoclonal. A single peak ofreactivity was found (FIG. 7) for a linear domain comprising the regioncontaining amino acids 142-158 (FIG. 8).

A comparison of the amino acid sequences of p24 of an HIV-1 isolate, p26of an HIV-2 isolate and p27 of SIV_(MAC) revealed conservation of adecapeptide (FIG. 9) within the epitope of p24 consisting ofSer-Pro-Arg-Thr-Leu-Asn-Ala-Trp-Val-Lys. It can be inferred that theregion encompassing the decapeptide is the 7-D4 epitope of p26 in HIV-2and p27 in SIV_(MAC).

The values of a defined epitope are known to those skilled in the art.One of the benefits is the ability of generating new antibodies capableof reacting with said epitope and similar epitopes. Synthetic peptidesare configured after the epitope sequence and either unmodified orconjugated to carriers are used as antigen. For example, peptides can beconjugated to PPD, tetanus toxoid, KLH or BSA using glutaraldehyde,carbodiimide or N-maleimidobenzoyl hydroxuccinimide ester. For a reviewof using synthetic peptides as antigen, see Ciba Foundation Symposium119 (1986) John Wiley and Sons, N.Y. Antibodies may be raised in vivo asin mice, goats or other lab animals or in vitro using a system ofmaterials and methods similar to the IVIS of Hana Biologics (Alameda,Calif). Another benefit is that large quantities of the epitope sequencecan be produced synthetically or using standard recombinant DNAtechniques as described above and the peptides can serve as antigen inimmunology-based assays and kits for the detection of circulatingantibody or for the detection of circulating antigen in an inhibitiontype assay. Another benefit relates to improving the assays disclosedherein. Without extending the survey, it is unclear whether the epitopeidentified in the HIV-1 isolate described herein is specific to thatisolate and furthermore to the HIV-2 and SIV isolates described herein.Using that sequence as a reference point, the epitope can be engineered,that is substituting one or more amino acids or alternativelyderivitizing the epitope, etc., with a view to identifying a relatedsequence with a greater degree of conservation among a larger variety ofHIV isolates or to obtaining a related sequence with a greater degree ofreactivity in assays. Although the nonapeptide analysis apparentlyidentified a discrete linear epitope comprised of amino acids 142-158 ofthe HIV-1 gag that is conserved in HIV-2 and SIV, it is to be understoodthat the instant invention relates to monoclonal antibodies, epitopes ofsaid monoclonal antibodies and assays using said antibodies and saidpeptides that are capable of detecting gag encoded proteins of HIV-1,HIV-2 and SIV.

Capture ELISA Assay

To determine which of the monoclonals would find utility in an ELISA,each was used as a capture or HRP-conjugate antibody in a sandwichassay. Briefly, the monoclonal antibody was coated on wells and 10 μl ofdisruption buffer added. The antigen samples suspended in detergentbuffer or controls in a volume of 100 μl were added next and incubatedat 37° C. for 90 minutes. After washing, bound antigen was detected byadding to the wells an enzyme conjugated anti-HIV reagent (horseradishperoxidase-conjugated human anti-HIV IgG, affinity purified, 100 μl) andincubated at 37° C. for 30 minutes. After washing several times, 100 μlof substrate solution were added to the wells and incubated at roomtemperature for 30 minutes. 100 μl of stop reagent were added andabsorbance read at 450 nm using an air blank. Representative data arepresented in Table 5.

                  TABLE 5    ______________________________________    Checkerboard Analysis of mAbs    Capture    Antibody           5B4    5D9    5E2  6C10 6E11 7E10 9B7  HαHIV    ______________________________________    5B4    0.12   0.26   0.29 0.82 0.13 1.03 0.17 2.67    5D9    0.73   0.13   0.43 0.62 0.37 0.38 0.12 >3.0    5E2    0.58   0.47   0.14 0.61 0.23 0.80 0.11 2.51    6C10   0.81   0.38   0.44 0.20 0.17 0.70 0.13 >3.0    6E11   0.09   0.21   0.21 0.14 0.16 0.27 0.09 0.41    7E10   0.84   0.43   0.49 0.84 0.18 0.18 0.13 >3.0    9B7    0.14   0.11   0.10 0.17 0.13 0.17 0.13 0.28    34A    0.49   0.12   0.08 0.96 0.28 1.81 0.22 >3.0    ______________________________________     Purified mAb were coated overnight at 10 μg/ml. HRPmAb used at 10     μg/ml added at beginning of incubation (90' at 37° C.).     HRPhuman-anti-HIV was added after 60 min.     Absorbances given for 10.0 ng/ml HIV1 MOLT 3 in NHS.     Absorbance for NHS was 0.12 ± 0.03

Antibodies 5-B4, 6-C10 and 7-E10 worked best as both capture andconjugated antibodies. Maximal signals were obtained with the HRP-humananti-HIV as the conjugate.

Various combinations of the monoclonals were used as capture antibodiesin ELISA's. The combination of 5-B4 and 6-C10 showed the greatestsensitivity in detecting p24 (FIG. 10). To detect p26 of HIV-2, 7-D4 wasused as a capture antibody (FIG. 11). It was found that maximalsensitivity and robustness occurred when the three antibodies, 5-B4,6-C10 and 7-D4 were combined as capture antibodies. Under thoseconditions, p26 was detectable as well as p24 from certain borderlineclinical samples that were difficult to interpret when only 5-B4 and6-C10 were used as capture antibodies. The sensitivity of the captureELISA using these three antibodies is less than 10 pg/ml (less than 1pg/well) of HIV-1 p24 antigen and less than 0.5 ng/ml of HIV-2 p26antigen (FIG. 12). The sensitivity is found despite the presence of HIVantibodies in the clinical samples. A capture ELISA using the threeantibodies 5-B4, 6-C10 and 7-D4 was also compared to a reversetranscriptase assay for the detection of whole virus. The ELISA was25,000 times more sensitive than the reverse transcriptase assay (FIG.13).

While the invention has been disclosed in this patent application byreference to the details of preferred embodiments of the invention, itis to be understood that this disclosure is intended in an illustrativerather than in a limiting sense, as it is contemplated thatmodifications will readily occur to those skilled in the art, within thespirit of the invention and the scope of the appended claims.

References

1. Clavel, F., et al., Science 233, 343 (1986)

2. Coates, A., et al., Nature 326, 549 (1987)

3. Guyader, M., et al., Nature 326, 662 (1987)

4. Kessler, H., et al., J Am Med Assoc 258, 1196 (1987)

5. Kohler, G, & Milstein, C., at Nature 256, 495 (1975)

6. Kuhnel, H., et al., Proc Natl Acad Sci USA 86, 2383 (1989)

7. Marlink, R., et al., AIDS Res Hum Retroviruses 4, 137 (1988)

8. Minassian, A., et al., Proc Natl Acad Sci USA 85, 6939 (1988)

9. Meissner, P. S. et al., Proc Natl Acad Sci USA 84, 4171 (1987)

10. Niedrig, M., et al., J Gen Virol 69, 2109 (1988)

11. Robert-Guroff, R. C. et al., Science 215, 975 (1982)

12. Sarngadharan, M. G., et al., Science 224, 506 (1984)

13. Starcich, B. R., et al., Cell 45, 637 (1986)

14. Towbin, H. et al., Proc Natl Acad Sci USA 76, 4350 (1979)

15. Wall, R., et al., Lancet i, 566 (1987)

16. Weiss, R., et al., AIDS 2, 95 (1988)

What is claimed is:
 1. A monoclonal antibody that cross-reacts with anepitope of p24 of HIV-1 and p26 of HIV-2, said epitope located withinamino acid residues 142-158 of p24 based on the numbering depicted inFIG.
 9. 2. A diagnostic kit for the detection of HIV-1 and HIV-2,comprising:(1) a container containing the monoclonal antibody of claim1; and (2) a container containing a labeled anti-HIV antibody that candetect immunocomplexes of the monoclonal antibody and the antigen of atleast one of HIV-1 and HIV-2.
 3. The diagnostic kit of claim 1, furthercomprising an additional monoclonal antibody that reacts with an antigenof HIV-1, wherein said additional monoclonal antibody does not reactwith said epitope of claim
 1. 4. The diagnostic kit of claim 3, whereinsaid additional monoclonal antibody that reacts with an antigen of HIV-1binds to an epitope located within amino acid residues 263-344 of p24.5. A method for detection of HIV-1 and HIV-₂ antigens in a sample,comprising contacting said sample with the monoclonal antibody of claim1, and measuring the formation of antigen-antibody complexes.
 6. Themethod of claim 5, further comprising contacting the sample with anadditional monoclonal antibody that has reactivity with an epitope ofHIV-1 other than the epitope of the monoclonal antibody of claim 5,prior to measuring the formation of antigen-antibody complexes.
 7. Themethod of claim 6, wherein the additional monoclonal antibody binds withan epitope located within amino acid residues 263-344 of p24, based onthe numbering depicted in FIG.
 5. 8. A method for detection of HIV-1 andHIV-2 antigens in a sample, which comprises contacting said sample withthe monoclonal antibody of claim 1, and an additional antibody thatreacts with an antigen of HIV-1 or HIV-2 but does not react with theepitope to which the monoclonal antibody of claim 1 reacts, andmeasuring the formation of antigen-antibody complexes.
 9. A monoclonalantibody according to claim 1, wherein said monoclonal antibody is 7-D4.10. A cell line for producing the monoclonal antibody according to claim9, having ATCC Accession Number HB
 11254. 11. The diagnostic kit ofclaim 2, wherein said monoclonal antibody is monoclonal antibody 7-D4.12. The method of claim 8, comprising contacting the sample with themonoclonal antibody 7-D4.